Containing anti-PLA2R IgG antibody induces podocyte injury in idiopathic membranous nephropathy

Abstract Background Idiopathic membranous nephropathy is widely recognized as an autoimmune kidney disease that is accompanied by the discovery of several autoantibodies, and the antibody subclass in the circulation of patients with iMN is mainly IgG. However, the direct pathogenic effect of the containing anti-PLA2R IgG antibody on podocytes is not clear. Method A protein G affinity chromatography column was used to purify serum IgG antibodies. Containing anti-PLA2R IgG antibodies from iMN patients and IgG from healthy controls were also obtained. Based on the established in vitro podocyte culture system, purified IgG antibodies from the two groups were used to stimulate podocytes, and the expression of essential podocyte proteins (podocin), the levels of inflammatory cytokines in the cell supernatant, cytoskeletal disorders, and podocyte apoptosis were analyzed. Results Compared with that in the normal IgG group, the expression of podocin and podocin mRNA was reduced (p = 0.016 and p = 0.005, respectively), the fluorescence intensity of podocin on the surface of podocytes was reduced, the cytoskeleton of podocytes was disordered and reorganized, and the ratio of podocyte apoptosis was increased in the iMN group (p = 0.008). Conclusion The containing anti-PLA2R IgG antibody might have a direct damaging effect on podocytes in idiopathic membranous nephropathy.

The discovery of anti-PLA2R antibodies has great clinical significance for the diagnosis and treatment of iMN.Studies have shown that the level of anti-PLA2R antibodies in peripheral blood is closely related to proteinuria remission in iMN patients [13][14][15] , and these findings suggest that anti-PLA2R antibodies may be involved in the occurrence and development of iMN.The antibody subclass in the circulation of patients with iMN is mainly IgG.However, the direct pathogenic effect of the containing anti-PLA2R IgG antibody on podocytes is not clear.
Podocytes are the innate cells in the glomerulus.They are mainly involved in forming the glomerular filtration barrier, maintaining the normal opening of capillary loops, synthesizing the basement membrane matrix, secreting cytokines, and regulating endothelial cell functions [16,17].Podocyte damage is a key factor in the progression of many diseases [18].Podocyte injury generally involves foot process fusion, a decrease in the number of podocytes, and apoptosis, which leads to the destruction of the glomerular filtration barrier and high levels of proteinuria [19].Foot process skeleton protein reorganization is an important cause of foot process fusion [20].Whether containing anti-PLA2R antibodies can cause podocyte damage in patients with iMN needs to be investigated.
To further clarify the role of IgG autoantibody in the pathogenesis of iMN, containing anti-PLA2R IgG antibodies were purified to determine the direct damaging effect on podocytes in vitro.

Purification of containing anti-PLA2R IgG autoantibody in vitro
Blood samples were collected from seven patients who were pathologically diagnosed with iMN, did not use immunosuppressants or hormones, had anti-PLA2R antibodies> 20 RU/ml and were anti-THSD7A antibody-negative on the day of admission.Blood samples from 4 healthy volunteers served as normal controls.The blood samples were stored at −80 °C.Containing anti-PLA2R IgG antibodies were purified from the sera of the seven iMN patients and four healthy controls using a HiTrap Protein G column (Swedish) on an AKTA-FPLC system(GE Biosciences, South San Francisco, CA, USA).

Cell culture
Human podocytes were provided by Prof. FanYi (Qilu Hospital, Cheeloo College of Medicine, Shandong University) and were cultured at 37 °C, centrifuged at 1000 rpm for 3 min, and discarded, and 10% fetal bovine serum in RPMI 1640 culture solution was added.After being cultured in a 33 °C incubator with 5% CO 2 , the cells were transferred to a 37 °C incubator for differentiation for 2 weeks.Before the experiment, the cells were incubated with serum-free RPMI 1640 medium for 12 h.
The experimental groups included the iMN containing anti-PLA2R IgG antibody group (iMN IgG), healthy human IgG (normal IgG) group, and blank control group (control).According to the time-response curve in vitro (Supplemental Figure ), containing anti-PLA2R IgG antibodies(200 µg/ml) and healthy human IgG (200 µg/ml) was added and incubated for 72h at 37 °C, 8% healthy serum was added to each group, three wells were set up, and the experiment was repeated three times.

Analysis of VEGF-a and TGF-β1 levels
After 72 h of incubation, podocyte supernatant was collected, and VEGF-A and TGF-β1 levels were analyzed using commercial ELISA kits (Elabscience, China).

Western blot analysis of purified containing anti-PLA2R IgG antibodies and podocyte-specific podocin
Western blotting was used to show that the purified serum IgG contained anti-PLA2R antibodies.Briefly, purified IgG from iMN patients was separated by 10% SDS-PAGE for 2 h and transferred to a polyvinylidene fluoride membrane for 2h.Target bands were incubated with rabbit anti-PLA2R1 antibodies (Sigma).Total podocyte proteins were extracted after the cells were incubated for 72 h with purified IgG.Podocyte lysate was separated by 5-10% SDS-PAGE for 2 h and transferred to a polyvinylidene fluoride membrane for 2h.Target bands were incubated with rabbit anti-podocin and rabbit anti-GAPDH antibodies overnight at 4 °C.After being washed, the target bands were incubated with horseradish peroxidase-conjugated IgG, and the blots were visualized with autoradiographic film using electro chemiluminescence plus western blotting detection system (GE Healthcare, Piscataway, USA).

Real-time quantitative PCR analysis of podocin
Total RNA was extracted from podocyte samples using an RNA isolation kit according to the manufacturer's instructions and reverse transcribed using a reverse transcriptase cDNA synthesis kit.Podocyte expression of podocin was examined using the SYBR Green PCR Master Mix assay with the following thermal cycling conditions: 95 °C for 5 s, followed by 40 cycles of 5 s at 95 °C and 34 s at 60 °C.Relative gene expression levels were calculated as 2 -ΔΔCt using GAPDH as the reference gene.

Immunofluorescence staining of podocin and the cytoskeleton
The cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 5 min.The cells were then stained with anti-podocin antibodies overnight or FITC-labeled phalloidin for 1 h.After being washed, the phalloidin-stained cells were imaged using an inverted fluorescence microscope (LEICA, Germany).TRITC-labeled goat anti-rabbit IgG was diluted in PBS and added to the anti-podocin antibody-stained cells.The stained samples were examined using an inverted fluorescence microscope.

Podocyte apoptosis detection
The percentage of apoptotic cells in the different groups was detected using an Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer's instructions.Briefly, different groups of podocytes were collected and suspended in a binding buffer after being centrifuged.The cells were then stained with annexin V-FITC for 5 min and with propidium iodide (PI) for 5 min in the dark.A control tube without annexin V-APC/PI was also prepared.The percentage of apoptotic podocytes was determined by flow cytometry (BD Biosciences).

Statistical analysis
For data with a nonnormal distribution, the results are expressed as the median (interquartile range, IQR).For normally distributed data, the results are expressed as the mean ± standard deviation (SD).Differences were assessed using a one-way analysis of variance (ANOVA) followed by Tukey's post hoc analysis or Mann-Whitney U tests as indicated.Statistical significance was set at p < 0.05.Analyses were performed using the SPSS statistical software package (ver.24.0).

The effect of containing anti-PLA2R IgG on the expression of VEGF-a and TGF-β1
Before performing the podocyte assay, purified IgG antibodies were shown to contain anti-PLA2R antibodies by western blotting (Figure 1a).VEGF-A and TGF-β1 are common inflammatory cytokines released by damaged cells, and the levels of these factors in the supernatant were determined by ELISA.After 72 h of incubation, the supernatant was collected for analysis.There were no significant differences in VEGF-A levels between containing anti-PLA2R IgG from iMN patients and normal IgG from healthy controls(832.86± 162.54 versus 794.78 ± 135.7 pg/ml, p = 0.758) (Figure 1b).Similarly, there was no significant difference in the levels of TGF-β1 between the two groups (4.51 ± 0.28 versus 4.76 ± 0.43 ng/ml, p = 0.371) (Figure 1c).

Effect on the expression of podocin
Since podocin lesions are involved in the pathogenesis of several podocytopathies, we used western blotting to measure the expression of podocin in podocytes.The results showed that the protein expression of podocin in the containing anti-PLA2RIgG group that was treated for 72h was lower than that in the normal IgG group (MFI: median 0.45, IQR 0.27-0.69versus median 0.97, IQR 0.52-1.61,p = 0.016) (Figure 2).

Effect on the expression of podocin mRNA
The mRNA expression of podocin in podocytes was detected by RT-PCR.The results showed that the mRNA expression of podocin in containing anti-PLA2R IgG group that was treated for 72 h was significantly lower than that in the normal IgG group (median 0.49, IQR 0.25-0.79versus median 1.11 IQR 1.01-1.14,p= 0.005) (Figure 3).

Effect on the expression of podocin, as determined by immunofluorescence assays
Podocin expression in podocytes was also detected by indirect immunofluorescence analysis.The results showed that the fluorescence intensity of podocin in containing the anti-PLA2R IgG group was weaker than that in the normal IgG group (Figure 4).

Effect of containing anti-PLA2R IgG on the morphology of the podocyte cytoskeleton
Cytoskeleton protein expression was detected by the direct immunofluorescence analysis.F-actin staining by phalloidin  1a shows purified igG containing the anti-Pla2R antibody, Figure 1b shows the expression level of VeGF-a in podocytes, and the right panel in Figure 1c shows the expression level of TGFβ1 in podocytes.There was no significant difference in the expression of VeGF-a (P = 0.758) and TGFβ1 (P = 0.371) between the containing anti-Pla2R igG group and the normal igG group.
showed obvious cytoskeleton disorders; the F-actin fibers were shortened, and the arrangement of the intracellular fibers was disorganized after stimulation with anti-PLA2R IgG (Figure 5).

Effect of containing anti-PLA2R IgG on podocyte apoptosis
Flow cytometry was used to detect podocyte apoptosis following IgG stimulation.The results showed that the proportion of podocyte apoptosis in the containing anti-PLA2R IgG group was higher than that in the normal IgG group (p = 0.008) (Figure 6).

Discussion
In this study, we observed that purified containing anti-PLA2R IgG antibodies from iMN patients could reduce the expression of podocin on the surface of podocytes, induce disorder and reorganization of the cytoskeleton, and increase podocyte apoptosis.These findings indicated that containing anti-PLA2R IgG antibodies may cause direct damage to podocytes, which may be independent of the complement system and may not be dependent on the PLA2 receptor on the surface of podocytes.These results provide a new understanding of the pathogenesis of iMN.
Anti-PLA2R antibodies are thought to be a milestone discovery in the pathogenesis of iMN in the past 10 years.Some studies have focused on whether these antibodies have a direct pathogenic role in primary membranous nephropathy.Li Y et al. found that purified anti-PLA2R antibodies could induce podocyte damage independent of the complement system.George et al. found that IgG4 targeted to PLA2R was aberrantly glycosylated and could activate the lectin pathway and induce podocyte injury [21,22].Lu [23] found that the integrity of the podocyte cytoskeletal structure was damaged, cell proliferation was decreased, and the apoptosis rate was increased by culturing podocytes with iMN patient serum.Skoberne et al. [24] also showed that patient serum containing anti-PLA2R antibodies interfered with the ability of podocytes to adhere to type IV collagen.However, the damaging effect of purified IgG derived from iMN patients on podocytes was not reported in vitro.
The cytoskeleton is a network of protein fibers in eukaryotic cells.Cytoskeleton remodeling is the basis for the increase in albumin permeability [25,26].Our experiments confirmed that when human podocytes were exposed to anti-PLA2R IgG antibodies, the cytoskeleton was reorganized.Previous studies have reported that the Rho/ROCK pathway is important for the regulation of cytoskeleton proteins.The integrin/FAK/PI3K pathway is also involved in actin adhesion to the basement membrane [27,28].We tried to clarify whether containing anti-PLA2R IgG antibody damaged podocytes directly in this study, and which signal pathways are involved in cytoskeletal rearrangement disorder requires further exploration.
The podocyte split membrane is composed of multiple proteins, including WT1, nephrin, podocin, Neph1, CD2AP, TRPC6, and BKca [29][30][31].We selected podocin as the target  protein and NPHS2 as the target gene.Our study that after stimulation with IgG extracted from iMN patients, human podocytes and podocin gene and protein expression were significantly decreased.In a study on diabetic nephropathy, serum podocin expression was reduced, and the expression of podocin was significantly inhibited in rat glomeruli stimulated by high glucose [32].In a study of patients with FSGS, it was found that the expression of podocin in kidney tissue was significantly lower than that in the normal population [33].Therefore, podocin is involved in the pathogenesis of diabetic nephropathy and FSGS.In a study of iMN, it was found that the decrease in podocin expression was negatively correlated with urine protein levels [34].In a study of the Heymann nephritis model, the protein expression of podocin decreased, and the decrease in podocin mRNA expression occurred earlier than the decrease in protein expression [35].In this study, we also observed that podocin protein and mRNA expression decreased after stimulation with IgG derived from iMN patients.
A decrease in the number of podocytes is an important part of the development of MN, and this process can be induced by apoptosis [36,37].Studies have shown that the Toll-like receptor 4 (TLR-4)/P2-7 pathway is involved in miR-186-mediated apoptosis [36].The miR-217/TLR4 pathway can improve podocyte apoptosis by downregulating XIST 37 .The expression of PLA2R in podocytes also participates in the pathogenesis of apoptosis.Yang et al. found that tissue PLA2R staining positively correlated with staining for apoptosis.Dongwei Liu [38] et al. further confirmed that PLA2R overexpression could induce podocyte apoptosis by downregulating the expression of miR-130a-5pin animal experiments.To date, there have only been a few studies on the relationship between containing anti-PLA2R antibodies and  cell apoptosis.This study showed that purified containing anti-PLA2R from iMN patients could induce podocyte In CKD, transforming growth factor-β (TGF-β) is a well-known cytokine that promotes fibrosis and induces podocyte apoptosis [39][40][41].Vascular endothelial growth factor A (VEGF-A) is a highly specific pro-vascular endothelial growth factor that is produced and secreted by podocytes and can promote endothelial cell proliferation, migration, and survival [42].Knockout of podocyte VEGF-A can damage the glomerular filtration barrier, leading to proteinuria and acute renal failure [43].Overexpression of VEGF-A in adult mice can induce abnormal proteinuria and kidney disease, affecting the structure and function of the glomerular barrier [44].An increase in VEGF-A overexpression leads to the disappearance of podocytes, the loss of the slit diaphragm, and eventually proteinuria [44,45].However, the expression of VEGF-A in iMN patients has rarely been reported.In our study, we found that the expression of TGF-β and VEGF-A did not change significantly after containing anti-PLA2R IgG stimulation of podocytes in iMN patients.These findings suggest that inflammatory cytokines may not act as the core effectors of the development of podocyte lesions.
In summary, we used purified anti-PLA2R IgG antibodies from iMN patients to stimulate podocytes and found that they could cause disorder of the podocyte skeleton, downregulate podocin expression, and directly induce apoptosis in podocytes.Whether the containing anti-PLA2R IgG antibodies include other unknown pathogenic factors or interact with the complement pathway to participate in the pathogenesis of iMN requires further investigation.

Funding
The National Natural Foundation of China (81600552 to Na Zhao) and Natural Science Foundation of Shandong Province (ZR2021MH282 to Na Zhao).

Figure 1 .
Figure 1.Expression of VEGF-a and TGFβ in the supernatant of podocytes.The left panel in Figure1ashows purified igG containing the anti-Pla2R antibody, Figure1bshows the expression level of VeGF-a in podocytes, and the right panel in Figure1cshows the expression level of TGFβ1 in podocytes.There was no significant difference in the expression of VeGF-a (P = 0.758) and TGFβ1 (P = 0.371) between the containing anti-Pla2R igG group and the normal igG group.

Figure 2 .
Figure 2. Podocin protein expression in podocytes.Containing anti-Pla2R igG group was stimulated for 72 h, and the expression of podocin was decreased compared with that in the normal igG stimulation group (P = 0.016).

Figure 3 .
Figure 3. Podocin mRNA expression in podocytes.The mRna expression of podocin in podocytes in the containing anti-Pla2R igG group was significantly lower than that in the normal igG stimulation group (P = 0.005).

Figure 4 .
Figure 4. Fluorescence intensity of podocin in podocytes.a1, a2 and a3 represent control groups; b1, b2 and b3 represent normal igG stimulation groups; and c1, c2 and c3 represent containing anti-Pla2R igG groups.The fluorescence intensity of podocin in the containing anti-Pla2R igG group was weaker than that in the normal igG group.

Figure 5 .
Figure 5. Immunofluorescence intensity of podocyte cytoskeletal proteins.a, b and c represent control group, normal igG stimulation group and containing anti-Pla2R igG group.The cytoskeleton fibers became shortened, and the arrangement of the fibers was disorganized in the iMn-igG-treated group.The cytoskeletal structure was disordered after stimulation of containing anti-Pla2R igG.